For several decades nitroglycerin has been administered to humans as a vasodilating agent in the treatment of cardiovascular disease. Recently, it has been shown that nitroglycerin so administered is converted in the body to nitric oxide which is the pharmacologically active metabolite. Still more recently, nitric oxide has been shown to be formed enzymatically from arginine as a normal metabolite which is an important component of endothelium-derived relaxing factors (EDRFs). EDRFs are currently being intensively studied as participating in regulation of blood flow and vascular resistance. In addition to vascular endothelium, macrophages have also been shown to produce nitric oxide in the body which is a component of their cell killing and/or cytostatic function.
More recently it has been established that the enzyme forming nitric oxide from arginine, i.e., nitric oxide synthase, occurs in two distinct forms, namely the constitutive form and the inducible form. The constitutive form is present in normal endothelial cells, neurons and some other tissues. Formation of nitric oxide by the constitutive form in endothelial cells is thought to play a role in normal blood pressure regulation. The inducible form of nitric oxide synthase has been found to be present in activated macrophages and is induced in endothelial cells and vascular smooth muscle cells, for example, by various cytokines and/or microbial products. It is thought that, in sepsis or cytokine-induced shock, overproduction of nitric oxide by the inducible form of nitric oxide synthase plays an important role in the observed life-threatening hypotension.
Considerable research effort has been expended to characterize the constitutive and inducible forms.
Thus far the constitutive form has been purified to homogeneity from rat and porcine cerebellum and rat neutrophils. Said purified constitutive form is reported to have a molecular mass as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis ranging from 150 kDa to 160 kDa, to appear to be a monomer, to require calcium and a calcium-binding protein such as calmodulin for its activation, and to be unstable (that isolated from rat cerebellum is reported to lose 50% of its enzyme activity when stored at 0.degree. C. for 2 hours). See Bredt, D. S., et al, Proc. Natl. Acad. Sci. USA, 87, 682-683 (1990); Mayer, B., et al, FEBS Lett., 277, 215-219 (1991); Schmidt, H. H. H. W., et al, Proced. Natl. Acad. Sci., USA 88, 365-369 (1991); Yui, Y , et al, J. Biol. Chem. 266, 3369-3371 (1991).
The induction of inducible form nitric oxide synthase activity from mouse macrophage cell line by exposure various cytokines and/or microbial products is reported in D. J., et al, J. Immunol. 139, 518-525 (1987); Drapier, J. C., et al, J. Immunol. 140, 2829-2838 (1988); and Ding. A. H., et al J. Immunol. 141, 2407-2412 (1988). Partial purification of activated mouse macrophage cell line inducible form nitric oxide synthase activity by affinity chromatography on adenosine 2', 5'-bisphosphate (2',5 ADP)-Sepharose resin is reported in Stuehr, D. J., et al, Biochem. Biophys. Res. Comm. Vol. 161, No. 2, 420-426 (6/15/89) and Kwon, S. K., et al, J. Biol. Chem. 264, 2049-20501 (1989) and Stuehr, D. J., et al, Biochem. Biophys. Res. Comm., Vol. 168, No. 2, 558-565 (4/30/90); by this method the inducible form activity is purified about 50-100-fold. For this partially purified inducible form nitric oxide synthase, nitric oxide synthesis was reported to be about 50% dependent on exogenous flavin adenine dinucleotide, about 50 % dependent on glutathione, 84% dependent on tetrahydrobiopterin, 95% dependent on NADPH and 98% dependent on L-arginine; see Stuehr, D. J., et al, Biochem. Biophys. Res. Comm. Vol. 168, No. 2, 558-565 (4/30/90). Purification of mouse macrophage cell line inducible form nitric oxide synthase activity 150-200-fold by FPLC anion exchange chromatography on a Mono Q column followed by affinity chromatography on 2',5.-ADP-Sepharose is reported in Stuehr, D. J., et al, J. Biol. Chem. Vol. 266, No. 10, 6259-6263 (4/5/91). Prior to the invention herein, there is no literature report of mouse macrophage cell line inducible form nitric oxide synthase activity having been purified to homogeneity.